At least 3 natural variants of hirudin are described in the literature (Markwardt 1970; Petersen et al. 1976; Markwardt and Walsmann 1976; Chang 1983; Dodt et al., 1984, 1985, 1986; Harvey et al. 1986; French Patent 84.04,755).
A comparison of the sequences of these 3 variants is shown in FIG. 1.
The first variant, HV1, corresponds to the hirudin which is isolated from the body of leeches; the second, HV2 (Harvey et al. 1986), differs from the first by 9 amino acids; the third (Dodt et al. 1986) is identical to HV2 as far as the serine 32, but differs by 10 amino acids in the C-terminal portion, which comprises, in addition, an additional amino acid (Ala63). This third variant will be designated hereinafter HV3.
These sequences contain 65 or 66 amino acids and may be regarded as 2 domains: a globular N-terminal portion, which contains 3 disulfide bridges, and an acidic C-terminal portion which possesses a homology with the site of cleavage by thrombin in the prothrombin molecule. This homology suggests that the region which surrounds position 47 might be the binding site of hirudin to thrombin.
Moreover, natural hirudin contains a sulfated tyrosine at position 63 (Chang, 1983). This sulfated tyrosine reappears at position 64 in the variant HV3. It will be designated hereinafter "position 63" for all the variants, its function being the same irrespective of its position.
A comparative analysis of the sequences of the natural variants of hirudin make it possible to envisage, theoretically, the creation of new variants in which the characteristics of the natural molecules are combined in different ways.
HV1 and HV3 have a lysine at position 47, situated between 2 prolines, which is probably responsible for blocking the active site of thrombin (Dodt et al. 1984 and 1985).
Since HV2 does not have a basic residue at this position but an asparagine, it seems desirable to substitute lysine or an arginine at this point in order to make the molecule more consistent with the features expected of a thrombin inhibitor (Chang 1985), or alternatively a histidine, which is not consistent with being a good substrate for thrombin but which might increase the inhibitory power of the hirudin.
Moreover, the sulfation of the tyrosine 63 represents a difference between natural hirudin and the hirudin obtained by genetic recombination which might have repercussions on its activity, as suggested by the kinetic studies of Stone and Hofsteenge (1986). An attempt may be made to mimic the native protein by replacing the tyrosine by an acidic residue such as Glu or Asp.